Highly Efficient Genetic Transformation Methods for the Marine Oleaginous Diatom Fistulifera solaris

The oleaginous diatom Fistulifera solaris is a promising producer of biofuel owing to the high content of the lipids. A genetic transformation technique by microparticle bombardment for this diatom was already established. However, the transformation efficiency was significantly lower than those of other diatoms. Devoting efforts to advance the genetic modifications of this diatom is crucial to unlock its full potential. In this study, we optimized the microparticle bombardment protocol, and newly established a multi-pulse electroporation protocol for this diatom. The nutrient-rich medium in the pre-culture stage played an essential role to increase the transformation efficiency of the bombardment method. On the other hand, use of the nutrient-rich medium in the electroporation experiments resulted in decreasing the efficiency because excess nutrient salts could hamper to establish the best conductivity condition. Adjustments on the number and voltage of the poring pulses were also critical to obtain the best balance between cell viability and efficient pore formation. Under the optimized conditions, the transformation efficiencies of microparticle bombardment and multi-pulse electroporation were 111 and 82 per 10(8) cells, respectively (37 and 27 times higher than the conventional bombardment method). With the aid of the optimized protocol, we successfully developed the transformant clone over-expressing the endogenous fat storage-inducing transmembrane protein (FIT)-like protein, which was previously found in the genome of the oleaginous diatom F. solaris and the oleaginous eustigmatophyte Nannochloropsis gaditana. This study provides powerful techniques to investigate and further enhance the metabolic functions of F. solaris by genetic engineering.